The value of electrophoresis in clinical chemistry has been recognized for some time in the analysis, for example, of proteins in serum as well as other body fluids. The use of electrophoresis in the clinical laboratory began in the early 1950s with the electrophoresis performed on paper strips. Proteins in, for example, serum were separated along a buffer-wetted strip of paper. After the separation was complete, the separated proteins were fixed and stained to produce visible patterns. These patterns were in the form of visible bands perpendicular to the direction of protein flow under the influence of the electrophoresizing voltage. In the interpretation of these electrophoresis patterns or bands, the relative intensities of the stained bands were examined to identify the proteins and the relative concentrations of the proteins within the sample.
The paper strips were later replaced with microporous membranes of either cellulose acetate or cellulose nitrate which provided better resolution due to the smaller pore size of these support materials. In addition to fixing and staining these membranes, the membranes could be "cleared" by soaking the membranes in a solvent, thereby collapsing the membrane pores without effecting the relative position of the stained bands.
With a microporous membrane thusly cleared, the membrane could be scanned by a scanning densitometer for quantitation of the visible protein bands. Such a scanning densitometer would use an illuminated slit and an optical detector on opposite sides of the membrane, generating a detected signal proprotional to the relative density of the protein bands. The resulting detected signal was displayed as a graph with density along the vertical axis and the relative gel position along the horizontal axis. The result was a representation of stain density and thus protein density along the membrane, with peaks along the graph corresponding to respective density maxima along the membrane. By comparing the areas under the peaks corresponding to the protein bands, a quantitative assessment of the relative distribution of the protein fractions contained in a sample could thus be made.
A next step in the development of clinical electrophoresis was the introduction of gel layers as the support media. A small quantity of the sample would be applied near one edge of a thin gel layer cast onto a stable backing material such as mylar. The gel would then be electrophoresized in a suitable electric field, processed by fixing and staining the separated protein fractions, and then viewed by examing the resulting stained gel directly or obtaining a quantitative scan of the gel in a scanning densitometer.
The interpretation of present clinical chemistry electrophoresis gels relies upon both the qualitative examination of the stained protein bands on the gel as well as the quantitative data of proportional protein content within the fractions indicated by the bands on the gel. Further, the gels may be incorporated as a part of a permanent medical record for later review, analysis or research. The gel patterns have thus become an important part of the pathologist's assessment of a patient's overall clinical condition in general and specific diagnostic interpretations in some instances.
Despite the success and acceptance of gel clinical electrophoresis, the technique requires skilled technicians and is time consuming, effectively limiting the number of tests that can be performed using the technique. Although automated gel electrophoresis analyzers are available, these analyzers are often very bulky and expensive, requiring considerable set-up time and effort.
Capillary electrophoresis is a more recent development and can be used to perform the type of electrophoretic separations presently performed with gels. In capillary electrophoresis, a small tube or capillary having an inside bore diameter in the range of about five microns to about two hundred microns and often about twenty cm long is filled with an electrically conductive fluid, or buffer. A small quantity of a sample to be analyzed is introduced into one end of the capillary bore and the ends of the capillary are placed into separate reservoirs of buffer. A direct current voltage in a range of about 2,000 volts to about 30,000 volts is applied to the ends of the capillary by means of electrodes positioned in the buffer reservoirs, causing a small current, typically in the range of about five microamps to about one milliamp, to flow through the capillary.
With the correct polarity applied across the capillary, the sample begins to migrate from the sample introduction end toward the other end of the capillary. As this migration occurs, different molecules in the sample travel at different rates primarily because of slightly different electrical charges on the molecules. These different migration rates cause molecules with slightly different charges to separate one from the other, some moving more quickly and advancing relatively with respect to more slowly moving molecules. As the sample nears the other end of the capillary, the small volume of sample becomes separated into bands of different molecules according to the relative migration rates of the molecules. These bands or groups of different molecules are detected near the other end of the capillary by, for example, passing a light beam through the bore of the capillary. Changes to the light beam, such as absorbance caused by the different molecules, are detected as the separated molecules pass through the beam, thus identifying the different molecules or the classes or categories of molecules in the sample and the relative concentration of such molecules.
Although automated forms of capillary electrophoresis analyzers are known in the art, none of such prior automated analyzers are suitable for routine clinical laboratory applications. The prior art analyzers require considerable manual manipulation despite their automated nature, allow only one sample to be electrophoresized at a time, have difficult capillary replacement procedures, or require additional external equipment such as high-pressure nitrogen tanks. In addition to these disadvantages, prior art capillary electrophoresis analyzers do not display the resulting electrophoretogram in a form that is readily acceptable to pathologists practicing in clinical chemistry laboratories. The failure of such prior analyzers to provide a display form or format that is usable by pathologists to evaluate the patient samples in a fashion consistent with gel electrophoresis represents a substantial and important drawback and disadvantage in such prior art systems with respect to the field of clinical chemistry.
Thus, there is a need for an automated capillary electrophoresis analyzer that provides a display form or format that is immediately and readily usable by pathologists in a fashion comparable to that obtained with gel electrophoresis. Further, there is a need for such an automated capillary electrophoresis analyzer that is easy to use, requires less sample manipulation, and is capable of substantially increased throughput as compared to prior capillary electrophoresis analyzers.